Methods for treating angelman syndrome and related disorders

ABSTRACT

Provided herein are methods of treating Angelman Syndrome and/or Prader-Willi syndrome, that include administering an effective amount of a T-type calcium channel antagonist to a subject in need of the treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/245,038, filed Oct. 22, 2015, the entire disclosure of which isincorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to treatment of disease by administeringpharmaceutical compounds. In particular, the disclosure relates to thetreatment of Angelman Syndrome and Prader-Willi syndrome byadministering a T-type calcium channel antagonist.

BACKGROUND

Angelman Syndrome is a genetic disorder caused by the deletion orinactivation of genes on the maternally inherited chromosome 15 withsilencing through imprinting of the paternal copy of the gene. Thesyndrome is characterized by intellectual and developmental disability,sleep disturbance, seizures, jerky movements, and a happy demeanor withfrequent laughter or smiling. Angelman Syndrome affects males andfemales in equal numbers, with a prevalence of about 1 in 15,000 of thegeneral population.

Angelman Syndrome is caused by the loss of the maternal contribution toa region of chromosome 15, most commonly by deletion althoughuniparental disomy, translocation, or single gene mutation in thatregion. In the region of the chromosome that is understood to becritical for Angelman Syndrome, the maternal and paternal contributionexpress certain genes very differently due to sex-specific epigeneticimprinting through DNA methylation. In a normal individual, the maternalallele of the gene UBE3A, part of the ubiquitin pathway, is expressedand the paternal allele is specifically silenced in the developingbrain. Angelman Syndrome results when the maternal contribution is lostor mutated. A related syndrome, Prader-Willi Syndrome, results if thepaternal contribution is lost or mutated.

The most common genetic defect leading to Angelman Syndrome is maternaldeletion of about 4 megabases in chromosomal region 15q11-13 causing anabsence of UBE3A expression in the paternally imprinted brain regions.UBE3A codes for an E6-AP ubiquitin ligase. Mice that do not expressmaternal UBE3A show severe impairments in hippocampal memory formationincluding a deficit in a learning paradigm that involveshippocampus-dependent contextual fear conditioning. Maintenance oflong-term synaptic plasticity in hippocampal area CA1 in vitro is alsodisrupted.

Symptoms of Angelman Syndrome include functionally severe developmentaldelay; speech impairment, with minimal or no use of words; movement andbalance disorders, including ataxia of gait and/or tremulous movement oflimbs, frequent laughter/smiling; apparent happy demeanor; easilyexcitable personality, often with hand flapping movements; hypermotoricbehavior; and short attention span. Frequently observed symptoms (in 80%or more of cases) include delayed, disproportionate growth in headcircumference, seizures and abnormal electroencephalogram (EEG). In theEEG, three distinct interictal patterns are seen in Angelman Syndrome,including a very large amplitude 2-3 Hz rhythm most prominent inprefrontal leads, a symmetrical 4-6 Hz high voltage rhythm, and a 3-6 Hzactivity punctuated by spikes and sharp waves in occipital leads, whichis associated with eye closure. In addition, symptoms associated withAngelman Syndrome, occurring in about 20 to 80% of cases, include flatocciput, occipital groove, protruding tongue, feeding problems,prognathia, wide mouth, drooling, mouthing behaviors, hypopigmentation,hyper active lower extremity deep tendon reflexes, increased sensitivityto head, abnormal sleep patterns, fascination with water, abnormal foodrelated behaviors, obesity, scoliosis, and constipation. Epilepsytypically occurs in 85% patients with Angelman Syndrome by age three,although development of seizures by age one occurs in less than 25% ofpatients. The types of seizures include atypical absences, generalizedtonic-clonic seizures, atonic seizures and myoclonic seizures. Somepatients experience multiple seizure types.

There is presently no cure available for Angelman Syndrome. SinceAngelman Syndrome can result in multiple varieties of seizures,selection of appropriate anticonvulsant medications to treat epilepsycan be difficult. Angelman Syndrome affects sleep patterns, so melatoninmay be used to promote sleep. Mild laxatives are also used frequently toencourage regular bowel movements. Beyond medication, physiotherapy isused to improve joint mobility and prevent stiffening of the joints.Speech and language therapy are commonly employed to addresscommunication issues.

T-type calcium channels are low-voltage activated calcium channels thatopen during membrane depolarization and mediate calcium influx intocells after an action potential or depolarizing signal. T-type calciumchannels known to be present within cardiac and smooth muscle, and alsoare present in many neuronal cells within the central nervous system.T-type calcium channels (transient opening calcium channels) aredistinct from L-type calcium channels (Long-Lasting calcium channels)due to their ability to be activated by more negative membranepotentials, their small single channel conductance, and theirnon-responsiveness to traditional calcium channel antagonist drugs,targeting L-type calcium channels.

T-type calcium channels open following small membrane depolarizations.T-type calcium channels have been primarily studied in the context ofneuronal and cardiomyocyte function, and have been implicated inhyperexcitability disorders, such as epilepsy and cardiac dysfunction.Voltage gated calcium channels are not generally expressed innon-excitable cells, but there is evidence that T-type calcium channelsare expressed in cancer cells of non-excitable lineages.

T-type calcium channels are activated and inactivated by small membranedepolarizations, and display slow deactivation rates. Thus, thesechannels can carry depolarizing current at low membrane potentials andmediate cellular “window” currents, which occur within the voltageoverlap between activation and steady state inactivation at low orresting membrane potentials. T-type calcium channels can maintain windowcurrent at non-stimulated or resting membrane potentials, therebyallowing a sustained inward calcium current carried by a portion ofchannels that are not inactivated. Mediation of window current allowsT-type calcium channels to regulate intracellular calcium levels, bothin electrically firing cells such as neurons, and in non-excitabletissues, under non-stimulated or resting cellular conditions.

Voltage-gated calcium channels are made up of several subunits. The α₁subunit is the primary subunit that forms the transmembrane pore of thechannel. The α₁ subunit also determines the type of calcium channel. Theβ, α₂δ, and γ subunits, present in only some types of calcium channels,are auxiliary subunits that play secondary roles in the channel. The alsubunit is composed of four domains (I-IV), with each domain containing6 transmembrane segments (S1-S6), and hydrophobic loops between the S5and S6 segments of each domain form the pore of the channel. Sub-typesof the T-type calcium channel are defined by the specific α₁ subunit asshown in Table 1.

TABLE 1 T-type Calcium Channel Sub-Types Designation α₁ subunit GeneCav3.1 α₁G CACNA1G Cav3.2 α₁H CACNA1H Cav3.3 α₁I CACNA1I

Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II or CaMKII) isa serine/threonine-specific protein kinase that is regulated by theCa′/calmodulin complex. CaMKII has 28 isoforms and is involved in manysignaling cascades and may be a mediator of learning and memory and mayplay a role in homeostasis and reuptake in cardiomyocytes, chloridetransport in epithelia, positive T-cell selection, and CD8 T-cellactivation. Misregulation of CaMKII may be linked to Alzheimer'sdisease, Angelman syndrome, and heart arrhythmia. CaMKII is alsoimplicated in long-term potentiation (LTP)—a molecular process ofstrengthening active synapses that may contribute to the processes ofmemory—and may therefore be important to memory formation.

The sensitivity of the CaMKII enzyme to calcium and calmodulin isgoverned by variable and self-associative domains. Initially, the enzymeis activated by binding to the calcium/calmodulin complex, which leadsto phosphorylation of the Threonine 286 site and activation of thecatalytic domain. Once activated, as greater amounts of calcium andcalmodulin accumulate, autophosphorylation can occur leading topersistent activation of the CaMKII enzyme. Autophosphorylation isenhanced by the stacked ring structure of the holoenzyme: the closeproximity of these rings allows for phosphorylation of neighboringCaMKII enzymes. Dephosphorylation of the Threonine 286 residue leads toinactivation of CaMKII.

SUMMARY

The present disclosure provides a method of treating Angelman Syndromeor Prader-Willi syndrome. The method includes administering to a subjectin need of such treatment a therapeutically effective amount of a T-typecalcium channel antagonist. Also provided is the use of -type calciumchannel antagonist for treating Angelman Syndrome or Prader-Willisyndrome. The disclosure also provides the use of -type calcium channelantagonist in the manufacture of a medicament for treating AngelmanSyndrome or Prader-Willi syndrome.

In some embodiments, the T-type calcium channel antagonist is a calciumchannel antagonist that selectively targets T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a smallmolecule.

In some embodiments, the T-type calcium channel antagonist is anantibody.

In some embodiments, the T-type calcium channel antagonist is a siRNA.

In some embodiments, the T-type calcium channel antagonist selectivelytargets CaV3.1.

In some embodiments, the T-type calcium channel antagonist selectivelytargets CaV3.2.

In some embodiments, the T-type calcium channel antagonist selectivelytargets CaV3.3.

In some embodiments, the T-type calcium channel antagonists antagonize aT-type calcium channel in a cell when the membrane potential of the cellis in the range from about −60 mV to about −30 mV, e.g., about −40 mV.

In some embodiments, T-type calcium channel antagonist is selected fromthe group consisting of mibefradil, diltiazem, nifedipine, nitrendipine,nimodipine, niludipine, niguldipine, nicardipine, nisoldipine,amlodipine, felodipine, isradipine, ryosidine, gallopamil, verapamil,tiapamil, pimozide, thioridazine, NNC 55-0396, TTL-1177, anandamide,pimozide, penfluridol, clopimozide, fluspirilene, haloperidol,droperidol, benperidol, triperidol, melperone, lenperone, azaperone,domperidone, antrafenine, aripiprazole, ciprofloxacin, dapiprazole,dropropizine, etoperidone, itraconazole, ketoconazole, levodropropizine,mepiprazole, naftopidil, nefazodone, niaprazine, oxypertine,posaconazole, trazodone, urpidil, vesnarinone, manidipine, nilvadipine,benidipine, efonidipine, flunarizine, anandamide, lomerizine, phenytoin,zonisamide, U-92032, tetralol, mibefradil, NNC 55-0396, TTA-A2, TTA-A8,TTA-P1, 4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3, TTA-Q6,MK-5395, MK-6526, MK-8998, Z941, Z944, ethosuximide, phensuximide,mesuximide, desmethylmethsuximide, efonidipine, trimethadione,dimethadione, ABT-639, TTL-1177, KYSO5044, nickel, and kurtoxin, andcombinations thereof.

In some embodiments, the T-type calcium channel antagonist is selectedfrom the group consisting of mibefradil, efonidipine, TTL-1177, andnickel, and combinations thereof.

In some embodiments, the T-type calcium channel antagonist ismibefradil.

In some embodiments, the T-type calcium channel antagonist is MK-8998.

In some embodiments, the disease is Angelman syndrome.

In some embodiments, the disease is Prader-Willi syndrome.

In some embodiments, the treatment comprises reducing or ameliorating aneurological symptom, which can be one or more of hyperactivity,abnormal sleep pattern, seizure, and ataxia.

In some embodiments, the treatment comprises reducing the frequency ofseizure in the subject.

In some embodiments, the treatment comprises reducing the severity ofseizure in the subject.

In some embodiments, the treatment comprises reducing the frequency ofdystonia in the subject.

In some embodiments, the treatment comprises reducing the severity ofdystonia in the subject.

In some embodiments, the treatment comprises reducing the frequency ofataxia in the subject.

In some embodiments, the treatment comprises reducing the severity ofataxia in the subject.

In some embodiments, the treatment comprises reducing the frequency oftremor, e.g., tremulous movement of the limbs, in the subject.

In some embodiments, the treatment comprises reducing the severity oftremor, e.g., tremulous movement of the limbs, in the subject.

In some embodiments, the treatment comprises reducing the severity ofataxia in the subject.

In some embodiments, the treatment comprises improving cognition orreducing cognitive deficits in the subject.

In some embodiments, the treatment comprises improving memory orreducing memory deficits in the subject.

In some embodiments, the treatment comprises improving attention orreducing attention deficits in the subject.

In some embodiments, the treatment comprises reducing obesity in thesubject.

In some embodiments, the treatment comprises reducing the amount or rateof weight gain in the subject.

In some embodiments, the treatment comprises reducing body weight in thesubject.

In some embodiments, the treatment comprises reducing hunger orincreasing satiety in the subject.

In some embodiments, the selective T-type calcium channel modulatorsubstantially crosses the blood brain barrier. In other embodiments, theT-type calcium channel modulator does not substantially cross the bloodbrain barrier.

In some embodiments, the treatment includes administering to the subjectan additional therapeutic agent, which can be, e.g., an additionalT-type calcium channel inhibitor or an anticonvulsive agent.

In some embodiments, the treatment includes administering an additionaltherapy, which can be selected, e.g., from the group consisting ofphysical therapy, occupational therapy, communication therapy, andbehavioral therapy.

The disclosure provides a method of treating a disease or disorderassociated with CaMKII autophosphorylation. The method includesadministering to a subject in need of such treatment a therapeuticallyeffective amount of a T-type calcium channel antagonist. Also providedis the use of -type calcium channel antagonist for treating a disease ordisorder associated with CaMKII autophosphorylation. The disclosure alsoprovides the use of -type calcium channel antagonist in the manufactureof a medicament for treating a disease or disorder associated withCaMKII autophosphorylation.

In some embodiments, the disease or disorder is Angelman Syndrome orPrader-Willi syndrome.

In some embodiments, the T-type calcium channel antagonist is a calciumchannel antagonist that selectively targets T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a smallmolecule.

In some embodiments, the T-type calcium channel antagonist is anantibody.

In some embodiments, the T-type calcium channel antagonist is a siRNA.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.1.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.2.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.3.

In some embodiments, the T-type calcium channel antagonists antagonize aT-type calcium channel in a cell when the membrane potential of the cellis in the range from about −60 mV to about −30 mV, e.g., about −40 mV.

Also provided is method of treating obesity in a subject in needthereof, comprising administering to the subject a T-type calciumchannel antagonist.

In some embodiments, the treatment comprises reducing body weight in thesubject.

In some embodiments, the treatment comprises reducing the amount or rateof weight gain in the subject.

In some embodiments, the treatment comprises reducing body weight in thesubject.

In some embodiments, the treatment comprises reducing hunger orincreasing satiety in the subject.

In some embodiments, the T-type calcium channel antagonist is a calciumchannel antagonist that selectively targets T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is a smallmolecule.

In some embodiments, the T-type calcium channel antagonist is anantibody.

In some embodiments, the T-type calcium channel antagonist is a siRNA.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.1.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.2.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.3.

In some embodiments, the T-type calcium channel antagonists antagonize aT-type calcium channel in a cell when the membrane potential of the cellis in the range from about −60 mV to about −30 mV, e.g., about −40 mV.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. Where the first page numberof a reference is given in a citation, it is to be understood thatreference is being made to the entire article cited. In case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of audiogenic-induced epilepsy tests usingUBE3/129sv mice treated with mibefradil (10 mg/kg, 40 mg/kg) or MK-8998(also known as “CX-8998”) (10 mg/kg, 30 mg/kg, 60 mg/kg).

DETAILED DESCRIPTION

The present disclosure describes that T-type voltage-gated calciumchannels are involved in Angelman Syndrome. The present disclosurefurther describes that modulation of such T-type voltage-gated calciumchannels can be effective for the treatment of Angelman Syndrome andrelated conditions such as Prader-Willi Syndrome.

While not being limited by any theory, a T-type calcium channelantagonist as described herein (e.g., mibefradil) can inhibit theinhibitory autophosphorylation of Ca²⁺/calmodulin-dependent proteinkinase II (i.e., CaMKII), which is a serine-threonine-specific proteinkinase that is regulated by the Ca²⁺/calmodulin complex and is involvedin various signaling cascades. Neuronal depolarization increases levelsof CaMKII autophosphorylation and chelation of extracellular calciumrobustly decreases basal CaMKII autophosphorylation and increases levelsof total CaMKII in cytosolic fractions. It has been shown thatinhibition of T-type calcium channels with mibefradil (5 μM) or NiCl₂(100 μM) significantly decreases threonine autophosphorylation ofCaMKIIα by 37% or 35% respectively, demonstrating that basal CaMKIIαactivation and autophosphorylation in the striatum is at least partiallysupported by calcium influx through T-type calcium channel inhibitors.Pasek et al., Mol. Cell. Neurosci., 2015, 68. 234-43.

Dysregulation of CaMKII is associated with neurological disordersincluding, but not limited to, Angelman Syndrome, Prader-Willi Syndrome,cerebral ischemia, and Alzheimer's disease, and it has been found thatmaintenance of basal levels of CaMKII autophosphorylation requiresT-type calcium channel activity. It is considered that that blockingT-type calcium channels can block the calcium from binding to CamKII,reducing inhibitory autophosphorylation and thus provide increasedactivation of CamKII and beneficial therapeutic effects.

Accordingly, the present application provides T-type calcium channelantagonists that can inhibit autophosphorylation of CaMKII and/orinhibit T-type calcium channels, which are useful for the treatment ofdisorders associated with dysregulation of CaMKII such as AngelmanSyndrome.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs.

For the terms “e.g.” and “such as,” and grammatical equivalents thereof,the phrase “and without limitation” is understood to follow unlessexplicitly stated otherwise.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

The term “about” means “approximately” (e.g., plus or minusapproximately 10% of the indicated value).

The term “small molecule” means an organic compound with a molecularweight of about 1000 or less.

The term “subject,” referring to the subject of treatment, means anyanimal, including mammals, e.g., human.

The phrase “therapeutically effective amount” refers to the amount ofactive compound or pharmaceutical agent that elicits the biological ormedicinal response that is being sought in a tissue, system, animal,individual or human by a researcher, veterinarian, medical doctor orother clinician.

The term “treating” or “treatment” refers to one or more of (1)preventing a disease; e.g., preventing a disease, condition or disorderin an individual who may be predisposed to the disease, condition ordisorder but does not yet experience or display the pathology orsymptomatology of the disease; (2) inhibiting a disease; e.g.,inhibiting a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., arresting further development ofthe pathology and/or symptomatology); and (3) ameliorating a disease;for example, ameliorating a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder (i.e., reversingthe pathology and/or symptomatology) such as decreasing the severity ofdisease or reducing or alleviating one or more symptoms of the disease.

The term “T-type calcium channel antagonists” refers to a substance thatreduces the activity of T-type calcium channels, e.g., through bindingto, or otherwise inhibiting or blocking activity of the channel, orthrough reducing the expression of T-type calcium channels.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, can also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, can also be provided separately or inany suitable subcombination.

The following abbreviations and symbols may be used in the presentdisclosure: CaMKII (calcium/calmodulin-dependent protein kinase II); DNA(deoxyribonucleic acid); dsRNA (double stranded RNA); g (gram); IC₅₀(half maximal inhibitory concentration); kg (kilogram); mg (milligram);mRNA (messenger RNA); RNA (ribonucleic acid); RNAi (RNA interference);siRNA (small interfering RNA), wt (weight).

II. Methods of Treatment

Provided herein are methods of treating a disease associated withdysregulation of CaMKII, dysregulation of T-type calcium channels, or acombination of dysregulation of CaMKII and dysregulation of T-typecalcium channels, in a subject in need thereof. The subject can includemice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, primates, and humans. In some embodiments, the subject is ahuman. In some embodiments, the treating comprises reducing orameliorating a neurological symptom associated with dysregulation ofCaMKII. In some embodiments, the neurological symptom is selected fromthe group consisting of mental retardation, cognitive dysfunction,deterioration of long-term potentiation (e.g., deterioration associatedwith Angelman Syndrome, Prader-Willi syndrome, or Alzheimer's disease),and ataxia. In some embodiments, the method comprises administering atherapeutically effective amount of a T-type calcium channel antagonistas described herein, or a pharmaceutically acceptable salt thereof, to asubject in need thereof to block inhibitory autophosphorylation ofCaMKII.

The present disclosure further provides methods of treating a diseaseselected from Angelman Syndrome and Prader-Willi syndrome in a subjectin need thereof. In some embodiments, the method comprises administeringa therapeutically effective amount of a T-type calcium channelantagonist as described herein, to the subject in need of the treatment.In some embodiments, the disease is Angelman Syndrome. In someembodiments, the disease is Prader-Willi syndrome.

In some embodiments, the treatment comprises reducing or ameliorating aneurological symptom associated with Angelman Syndrome or Prader-WilliSyndrome. The neurological symptoms can include but are not limited, toany one or more of hyperactivity, abnormal sleep pattern, seizure, andataxia. In some embodiments, the neurological symptom comprises seizure.As used herein, the term “seizure” includes, but is not limited to, andcan be any one or more of absence seizures (e.g., typical and atypicalabsences), atonic seizures, catamenial seizures, cluster seizures,episodic seizures, Dravet syndrome (i.e., severe myoclonic epilepsy ininfancy or SMEI), focal seizures (i.e., partial seizures), focalseizures with secondary generalization, focal seizures with secondarygeneralized clonic seizures, infantile spasmsmyoclonic seizures, tonicseizures, and tonic clonic seizures.

In some embodiments, the subject of treatment has symptoms that includehyperactivity. In some embodiments, the subject of treatment hassymptoms that include abnormal sleep pattern. In some embodiments, thesubject of treatment has symptoms that include seizure, including anyone or more of the types of seizure defined above. In some embodiments,the subject of treatment has symptoms that do not include ataxia. Insome embodiments, the subject of treatment has symptoms that do notinclude abnormal sleep pattern. In some embodiments, the subject oftreatment has symptoms that do not include seizure, or that do notinclude one or more of atypical absence seizures, typical absences,atonic seizures, catamenial seizures, cluster seizures, episodicseizures, Dravet syndrome focal seizures (with or without secondarygeneralization or secondary generalized clonic seizures), infantilespasms, myoclonic seizures, tonic seizures, or tonic clonic seizures.

Also provided is method of treating obesity in a subject in needthereof, comprising administering to the subject a T-type calciumchannel antagonist. In some embodiments, the treatment comprisesreducing body weight in the subject. In some embodiments, the treatmentcomprises reducing the amount or rate of weight gain in the subject. Insome embodiments, the treatment comprises reducing body weight in thesubject. In some embodiments, the treatment comprises reducing hunger orincreasing satiety in the subject.

The treatment can be administered at an effective dose for theparticular compound. Examples of suitable doses include, in humans,include dosages in the range from about 1 mg to about 2000 mg, e.g.,about 1 mg to about 2000 mg, about 2 mg to about 2000 mg, about 5 mg toabout 2000 mg, about 10 mg to about 2000 mg, about 20 mg to about 2000mg, about 50 mg to about 2000 mg, about 100 mg to about 2000 mg, about150 mg to about 2000 mg, about 200 mg to about 2000 mg, about 250 mg toabout 2000 mg, about 300 mg to about 2000 mg, about 400 mg to about 2000mg, about 500 mg to about 2000 mg, about 1000 mg to about 2000 mg, about1 mg to about 1000 mg, about 2 mg to about 1000 mg, about 5 mg to about1000 mg, about 10 mg to about 1000 mg, about 20 mg to about 1000 mg,about 50 mg to about 1000 mg, about 100 mg to about 1000 mg, about 150mg to about 1000 mg, about 200 mg to about 1000 mg, about 250 mg toabout 1000 mg, about 300 mg to about 1000 mg, about 400 mg to about 1000mg, about 500 mg to about 1000 mg, about 1 mg to about 500 mg, about 2mg to about 500 mg, about 5 mg to about 500 mg, about 10 mg to about 500mg, about 20 mg to about 500 mg, about 50 mg to about 500 mg, about 100mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about500 mg, about 1 mg to about 250 mg, about 2 mg to about 250 mg, about 5mg to about 250 mg, about 10 mg to about 250 mg, about 20 mg to about250 mg, about 50 mg to about 250 mg, about 100 mg to about 250 mg, about1 mg to about 100 mg, about 2 mg to about 100 mg, about 5 mg to about100 mg, about 10 mg to about 100 mg, about 20 mg to about 100 mg, about50 mg to about 100 mg. Doses can be, e.g., about 1 mg, about 2 mg, about5 mg, about 10 mg, about 20 mg, about 50 mg, about 100 mg, about 150 mg,about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg,about 1000 mg, about 1500 mg, or about 2000 mg. Doses can be less thanabout 2000 mg, less than about 1500 mg, less than about 1000 mg, lessthan about 5000 mg, less than about 400 mg, less than about 250 mg, lessthan about 200 mg, less than about 150 mg, less than about 100 mg, lessthan about 50 mg, less than about 20 mg or less than about 10 mg. Eachof the doses can be doses that are administered at a frequency of oncedaily, twice daily, three times daily or four times daily, or less thanonce daily. Each of the doses can also be the dose that is administeredto an adult with equivalent (scaled) dose being administered forpediatric patients.

The dose can be a dose that provides a plasma level (e.g., a steadystate or a maximum level) of about 100 ng/mL, about 200 ng/mL, 500ng/mL, about 1 μg/mL, about 2 μg/mL, about 5 μg/mL, about 10 μg/mL,about 20 μg/mL, about 50 μg/mL, about 100 μg/mL, about 200 μg/mL, about250 μg/mL, or about 500 μg/mL, about 1000 μg/mL, or in a range betweenthese values, or a concentration that is less than these values.

In some embodiments, treatment is continued for a period of about 1 weekor longer. In some embodiments, treatment is continued for a period ofabout 2 weeks or longer. In some embodiments, treatment is continued fora period of about 3 weeks or longer. In some embodiments, treatment iscontinued for a period of about 4 weeks or longer. In some embodiments,treatment is continued for a period of about 8 weeks or longer. In someembodiments, treatment is continued for a period of about 12 weeks, orabout 13 weeks, or longer. In some embodiments, treatment is continuedfor a period of about 24 weeks, or 26 weeks, or longer. In someembodiments, treatment is continued for a period of about 6 months orlonger. In some embodiments, treatment is continued for a period ofabout 12 months or longer. In some embodiments, treatment is continuedfor a period of about 18 months or longer. In some embodiments,treatment is continued for a period of about 24 months or longer.

III. T-Type Calcium Channel Antagonists

The T-type calcium channel antagonist used in any of the methodsdescribed herein, or any of the embodiments thereof, can be one or moreof the T-type calcium channel agonists described below.

The T-type calcium channel antagonist can be an antagonist of humanT-type calcium channels when the subject of treatment is a human.

The T-type calcium channel antagonist can be a small molecule. Examplesmall molecule T-type calcium channel antagonists which may be used inthe methods provided herein include, but are not limited to, mibefradil,diltiazem, nifedipine, nitrendipine, nimodipine, niludipine,niguldipine, nicardipine, nisoldipine, amlodipine, felodipine,isradipine, ryosidine, gallopamil, verapamil, tiapamil, pimozide,thioridazine, NNC 55-0396, TTL-1177, anandamide, benzazepinederivatives, diphenylbutylpiperidine derivatives (e.g., pimozide,penfluridol, clopimozide, and fluspirilene), butyrophenone derivatives(e.g., haloperidol, droperidol, benperidol, triperidol, melperone,lenperone, azaperone, and domperidone), and phenylpiperazine derivatives(e.g., antrafenine, aripiprazole, ciprofloxacin, dapiprazole,dropropizine, etoperidone, itraconazole, ketoconazole, levodropropizine,mepiprazole, naftopidil, nefazodone, niaprazine, oxypertine,posaconazole, trazodone, urpidil, and vesnarinone), dihydropyridinederivatives (e.g., manidipine, nilvadipine, benidipine, andefonidipine), flunarizine, anandamide, lomerizine, phenytoin,zonisamide, U-92032, tetralol, tetralol derivatives (e.g., mibefradil),mibefradil derivatives (e.g., NNC 55-0396 dihydrochloride), TTA-A2,TTA-A8, TTA-P1, 4-aminomethyl-4-fluoropiperidine (TTA-P2), TTA-Q3,TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, succinimideanticonvulsant derivatives (e.g., ethosuximide, phensuximide, andmesuximide also known as methsuximide, N-desmethylmethsuximide alsoknown as (alpha)-methyl-(alpha)-phenyl-succinimide), and efonidipine(e.g. (R)-efonidipine), trimethadione, dimethadione, ABT-639, TTL-1177,KYSO5044, kurtoxin. Any of the T-type calcium channel inhibitors can bein the form of a pharmaceutically acceptable salt. Structures of certainT-type calcium channel inhibitors are shown below:

In some embodiments, T-type calcium channel small-molecule modulatorsmay be selected from the group consisting of those described in thepatents and published patent applications listed in Giordanetto et al,“T-type calcium channels inhibitors: a patent review,” Expert Opin.Ther. Pat., 2011, 21, 85-101, including WO2004035000, WO9304047,WO2006098969, WO2009009015, WO2007002361, WO2007002884, WO2007120729,WO2009054982, WO2009054983, WO2009054984, US20090270413, WO2008110008,WO2009146539, WO2009146540, U.S. Pat. No. 8,133,998, WO2010083264,WO2006023881, WO2006023883, WO2005007124, WO2005009392, US2005245535,WO2007073497, WO200707852, WO2008033447, WO2008033456, WO2008033460,WO2008033464, WO2008033465, WO2008050200, WO20081 17148, WO2009056934,EP1568695, WO2008007835, KR754325, U.S. Pat. No. 7,319,098,US20100004286, EP1757590, KR2009044924, US2010094006, WO2009035307,US20090325979, KR75758317, WO2008018655, US20080293786, andUS20100056545, each of which is incorporated by reference in itsentirety.

In some embodiments, the T-type calcium channel antagonist is a smallmolecule. In some embodiments, the small molecule has a molecular weightof 1000 or lower, e.g., about 900 or lower, about 800 or lower, about700 or lower, about 600 or lower, about 500 or lower, about 400 orlower, or in the range from about 100 to about 500, about 200 to about500, about 200 to about 400, about 300 to about 400 or about 300 toabout 500. In some embodiments, the T-type calcium channel antagonist isa selective T-type calcium channel antagonist. “Selective” in thiscontext means that the T-type calcium channel antagonist is more potentat antagonizing T-type calcium channel calcium channels compared withother types of calcium channel, e.g., any one or more of L-type, N-type,P-type, Q-type and/or R-type calcium channels, e.g., compared withL-type calcium channels. Selectivity can be determined, e.g., bycomparing the IC₅₀ of a compound in inhibiting T-type calcium channelswith its IC₅₀ in inhibiting the other types of calcium channel: if theIC₅₀ for inhibiting T-type channels is lower than the IC₅₀ forinhibiting the other types of calcium channel, the compound isconsidered selective. An IC₅₀ ratio of 0.1 (or lower) denotes 10-fold(or greater) selectivity. An IC₅₀ ratio of 0.01 (or lower) denotes100-fold (or greater) selectivity. An IC₅₀ ratio of 0.001 (or lower)denotes 1000-fold (or greater) selectivity. In some embodiments, theT-type calcium channel antagonist has selectivity for the T-type calciumchannel that is 10-fold or greater, 100-fold or greater, or 1000-fold orgreater compared with other types of calcium channel, e.g., any one ormore of L-type, N-type, P-type, Q-type and/or R-type calcium channels,e.g., compared with L-type calcium channels.

In some embodiments, the T-type calcium channel antagonist is aselective T-type calcium channel inhibitor which is selected from thegroup consisting of phensuximide, methsuximide,methyl-phenyl-succinimide, R isomer of efonidipine, trimethadione,dimethadione, mibefradil, TTA-A2, TTA-A8, TTA-P1, TTA-P2, TTA-Q3,TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944, ABT-639, TTL-1177,KYSO5044, N C 55-0396 dihydrochloride, kurtoxin, or a derivativethereof.

In some embodiments, the T-type calcium channel antagonist is selectedfrom the group consisting of mibefradil, efonidipine, TTL-1177, nickel,and combinations thereof.

In some embodiments, the T-type calcium channel antagonist ismibefradil.

In some embodiments, the T-type calcium channel antagonist is MK-5395.

In some embodiments, the T-type calcium channel antagonist is MK-6526.

In some embodiments, the T-type calcium channel antagonist is MK-8998.

In some embodiments, the T-type calcium channel antagonist is Z944.

In some embodiments, the T-type calcium channel antagonist can be otherthan ethosuximide.

In some embodiments, the T-type calcium channel antagonist can be amolecule that does not act as a pore-blocker of the T-type calciumchannel. The T-type calcium channel antagonist can be, e.g., anallosteric inhibitor of T-type calcium channels.

In some embodiments, the T-type calcium channel antagonist can be onethat does not substantially affect one or more sodium channels such assodium channels having Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav1.6, Nav 1.7, Nav 1.8, or Nav 1.9 alpha subunits, and/or Nav β1, Nav β2,Nav β3, Nav β4 subunits. The T-type calcium channel antagonist can beselective for T-type calcium channel compared to inhibition of sodiumchannels, e.g., having at least a 2-fold, at least a 5-fold, at least a10-fold, at least a 20-fold, at least a 100-fold, at least a 500-fold orat least a 1000-fold selectivity (expressed, e.g., in terms of K_(i)).The T-type calcium channel inhibitor can be one that does notsubstantially decrease the non-inactivating sodium current inthalamocortical neurons, e.g., that decreases the inactivating sodiumcurrent by about 20% or less, about 10% or less, about 5% or less, about2% or less, or about 1% or less.

In some embodiments, the T-type calcium channel antagonist can be onethat does not substantially affect one or more potassium channels suchas calcium activated potassium channels (BK channels, SK channels, IKchannels), inwardly rectifying potassium channels (ROMK, GPCR regulated,ATP sensitive), tandem pore domain potassium channels (TWIK (TWIK-1,TWIK-2, KCNK7), TREK (TREK-1, TREK-2, TRAAK), TASK (TASK-1, TASK-3,TASK-5), TALK (TASK-2, TALK-1, TALK-2), THIK (THIK-1, THIK-2), TRESK),or voltage gated potassium channels (hERG, KvLQT). The T-type calciumchannel antagonist can be selective for T-type calcium channel comparedto inhibition of potassium channels, e.g., having at least a 2-fold, atleast a 5-fold, at least a 10-fold, at least a 20-fold, at least a100-fold, at least a 500-fold or at least a 1000-fold selectivity(expressed, e.g., in terms of K_(i)).

In some embodiments, the T-type calcium channel antagonist can be onethat does not substantially affect one or more GABA receptors such asGABA_(A) receptors, GABA_(A-ρ) subclass (GABA_(C)) receptors, orGABA_(B) receptors. In some embodiments, the T-type calcium channelantagonist can be one that does not substantially affect one or moresubunits of the GABA_(A) receptors such as α-subunits (GABRA1, GABRA2,GABRA3, GABRA4, GABRA5, GABRA6), β-subunits (GABRB1, GABRB2, GABRB3),γ-subunits (GABRG1, GABRG2, GABRG3), δ-subunits (GABRD), ε-subunits(GABRE), π-subunits (GABRP), θ-subunits (GABRQ), particularly GABARA5,GABRB3 and GABRG5. The T-type calcium channel antagonist can beselective for T-type calcium channel compared to inhibition of GABAreceptors, e.g., having at least a 2-fold, at least a 5-fold, at least a10-fold, at least a 20-fold, at least a 100-fold, at least a 500-fold orat least a 1000-fold selectivity (expressed, e.g., in terms of K_(i) orbinding affinity).

In some embodiments, the T-type calcium channel antagonist can be onethat does not cause one or more of the following side-effects or adverseevents upon administration to animals, e.g., humans: liver damage,morphological changes in the animal liver, functional changes in theanimal liver, kidney damage, morphological changes in the animal kidney,functional changes in the animal kidney, systemic lupus erythematosus,suicidal thoughts, suicidal behavior, suicidal ideation, increased riskof suicide, emergence or worsening of depression, unusual changes inmood or behavior, birth defects, allergic reaction.

In some embodiments, the T-type calcium channel antagonist can be onethat does not cause one or more of the following side-effects or adverseevents upon administration to animals: adverse events involving thegastrointestinal system such as anorexia, vague gastric upset, nauseaand vomiting, cramps, epigastric and abdominal pain, weight loss,diarrhea, gum hypertrophy and swelling of the tongue; adverse eventsinvolving the hemopoietic system such as leukopenia, agranulocytosis,pancytopenia, with or without bone marrow suppression, and eosinophilia;adverse events involving the nervous system, including neurologicalreactions, sensory reactions, or psychiatric or psychologicalaberrations such as drowsiness, headache, dizziness, euphoria, hiccups,irritability, hyperactivity, lethargy, fatigue, ataxia, confusion,disturbances of sleep, night terrors, inability to concentrate,aggressiveness, paranoid psychosis, increased libido, or increased stateof depression with overt suicidal intentions; adverse events involvingthe integumentary system including dermatologic manifestations such asurticaria, Stevens-Johnson syndrome, systemic lupus erythematosus,pruritic erythematous rashes, and hirsutism; adverse events involvingthe special senses such as myopia; and adverse events involving thegenitourinary system, such as vaginal bleeding or microscopic hematuria.

In some embodiments, the T-type calcium channel antagonist is anantibody. Various methods for the preparation of antibodies are known inthe art. See, Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow,and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY(1989). For example, antibodies can be prepared by immunizing a suitablemammalian host with a sample of whole cells isolated from a patient.Antibodies can be produced by cell culture techniques, including thegeneration of monoclonal antibodies as described herein, or viatransfection of antibody genes into suitable bacterial or mammalian cellhosts, in order to allow for the production of recombinant antibodies.

In some embodiments, the antibody is a monoclonal antibody. A“monoclonal antibody” is an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the antibodies comprisingthe population are identical except for possible naturally occurringmutations that are present in minor amounts.

In some embodiments, an antibody provided herein can be produced byrecombinant means. In some embodiments, the antibody is a “humanized” orhuman antibody. “Humanized” or human antibodies can also be produced,and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody sequences for corresponding humanantibody sequences, are well known. See, e.g., Jones et al., Nature,1986, 321, 522-25; Riechmann et al., Nature, 1988, 332, 323-27;Verhoeyen et al., Science, 1988, 239, 1534-36, Carter et al., Proc.Natl. Acad. Sci. USA, 1993, 89, 4285; and Sims et al., J. Immunol.,1993, 151, 2296. These humanized antibodies are designed to minimizeunwanted immunological response toward rodent antihuman antibodymolecules which limits the duration and effectiveness of therapeuticapplications of those moieties in human recipients. Accordingly,preferred antibodies used in the therapeutic methods described hereinare those that are either fully human or humanized with high affinitybut exhibit low or no antigenicity in the subject.

In some embodiments, the T-type calcium channel antagonist is anoligonucleotide inhibitor. Example oligonucleotide inhibitors include,but are not limited to, antisense oligonucleotides, RNAi, dsRNA, siRNAand ribozymes. In some embodiments, the T-type calcium channelantagonist is a siRNA. As used in the specification, “antisenseoligonucleotide” refers to a stretch of single-stranded DNA or RNA,usually chemically modified, whose sequence (3′-5′) is complementary tothe sense sequence of a molecule of mRNA. Antisense moleculeseffectively inhibit gene expression by forming RNA/DNA duplexes.Antisense is understood to work by a variety of mechanisms, includingphysically blocking the ability of ribosomes to move along the messengerRNA, and hastening the rate at which the mRNA is degraded within thecytosol.

In order to avoid digestion by DNAse, antisense oligonucleotides can bechemically modified. For example, phosphorothioate oligodeoxynucleotidesare stabilized to resist nuclease digestion by substituting one of thenon-bridging phosphoryl oxygen of DNA with a sulfur moiety. Increasedantisense oligonucleotide stability can also be achieved using moleculeswith 2-methoxyethyl (MOE) substituted backbones as described generallyin U.S. Pat. No. 6,451,991, incorporated by reference, and U.S. Pat.Appl. Publ. No. 2003/0158143-A1. Thus, the antisense oligonucleotide canbe modified to enhance in vivo stability relative to an unmodifiedoligonucleotide of the same sequence. The modification may be, e.g., a(2′-O-2-methoxyethyl) modification. The oligonucleotide may have aphosphorothioate backbone throughout, the sugar moieties of nucleotides1-4 and 18-21 may bear 2′-O-methoxyethyl modifications and the remainingnucleotides may be 2′-deoxynucleotides.

It is understood in the art that an antisense oligonucleotide need nothave 100% identity with the complement of its target sequence in orderto be effective. The antisense oligonucleotides, therefore, can have asequence that is at least about 70% identical to the complement of thetarget sequence. In one embodiment, the antisense oligonucleotides havecan a sequence that is at least about 80% identical to the complement ofthe target sequence. In other embodiments, they have a sequence that isat least about 90% identical or at least about 95% identical to thecomplement of the target sequence, allowing for gaps or mismatches ofseveral bases. Identity can be determined, for example, by using theBLASTN program of the University of Wisconsin Computer Group (GCG)software.

The antisense oligonucleotides according to the present invention aretypically between 7 and 100 nucleotides in length. In one embodiment,the antisense oligonucleotides comprise from about 7 to about 50nucleotides, or nucleotide analogues. In another embodiment, theantisense oligonucleotides comprise from about 7 to about 35nucleotides, or nucleotide analogues. In other embodiments, theantisense oligonucleotides comprise from about 12 to about 35nucleotides, or nucleotide analogues, and from about 15 to about 25nucleotides, or nucleotide analogues.

The oligonucleotide inhibitors according to the present invention can besiRNA molecules that are targeted to a gene of interest such that thesequence of the siRNA corresponds to a portion of said gene. RNAmolecules used in the present invention generally comprise an RNAportion and some additional portion, for example a deoxyribonucleotideportion.

The present disclosure further contemplates ribozyme oligonucleotidemodulators that specifically target mRNA encoding a protein of interest,such as the proteins comprising the T-type calcium channel. Ribozymesare RNA molecules having an enzymatic activity that enables the ribozymeto repeatedly cleave other separate RNA molecules in anucleotide-sequence specific manner. Such enzymatic RNA molecules can betargeted to virtually any mRNA transcript, and efficient cleavage can beachieved in vitro. Kim et al., Proc. Natl. Acad. Sci. USA, 1987, 84,8788; Haseloff et al., Nature, 1988, 334, 585; Cech, JAMA, 1988, 260,3030; and Jefferies et al., Nucleic Acids Res., 1989, 17, 1371.

Typically, a ribozyme comprises two portions held in close proximity: anmRNA binding portion having a sequence complementary to the target mRNAsequence and a catalytic portion which acts to cleave the target mRNA. Aribozyme acts by first recognizing and binding a target mRNA bycomplementary base-pairing through the target mRNA binding portion ofthe ribozyme. Once it is specifically bound to its target, the ribozymecatalyzes cleavage of the target mRNA. Such strategic cleavage destroysthe ability of a target mRNA to direct synthesis of an encoded protein.Having bound and cleaved its mRNA target, the ribozyme is released andcan repeatedly bind and cleave new target mRNA molecules.

In some embodiments, the selective T-type calcium channel antagonistsubstantially crosses the blood brain barrier.

In some embodiments, the selective T-type calcium channel antagonistdoes not substantially cross the blood brain barrier.

In some embodiments, the T-type calcium channel antagonist is a calciumchannel antagonist that selectively targets T-type calcium channels. Insome embodiments, the T-type calcium channel antagonist is a smallmolecule as described herein.

In some embodiments, the T-type calcium channel antagonist selectivelytargets Cav3.1. In some embodiments, the T-type calcium channelantagonist selectively targets Cav3.2. In some embodiments, the T-typecalcium channel antagonist selectively targets Cav3.3. “Selective” inthis context means that the T-type calcium channel antagonist is morepotent at antagonizing one type of T-type calcium channel over anothertype of calcium channel, e.g., more potent at antagonizing Cav3.1 thanCav3.2 or Cav3.3 or both; more potent at antagonizing Cav3.2 than Cav3.1or Cav3.3 or both; more potent at antagonizing Cav3.3 than Cav3.1 orCav3.2 or both. Selectivity can be determined, e.g., by comparing theIC₅₀ of a compound in inhibiting one type of T-type calcium channel withits IC₅₀ in inhibiting the other types of T-type calcium channel: if theIC₅₀ for inhibiting one type of T-type channels is lower than the IC₅₀for inhibiting the other type of T-type calcium channel, the compound isconsidered selective. An IC₅₀ ratio of 0.1 (or lower) denotes 10-fold(or greater) selectivity. An IC₅₀ ratio of 0.01 (or lower) denotes100-fold (or greater) selectivity. An IC₅₀ ratio of 0.001 (or lower)denotes 1000-fold (or greater) selectivity. In some embodiments, theselectivity for Cav3.1, Cav3.2 or Cav3.3 is 10-fold or greater, 100-foldor greater, or 1000-fold or greater.

In some embodiments, the T-type calcium channel antagonist selectivelytargets T-type calcium channels (e.g., Cav3.1, Cav3.2, and/or Cav3.3)over sodium channels such as sodium channels having Nav 1.1, Nav 1.2,Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, or Nav 1.9 alphasubunits, and/or Nav β1, Nav β2, Nav β3, Nav β4 subunits. The T-typecalcium channel antagonist can be selective for T-type calcium channelcompared to inhibition of sodium channels. Selectivity can bedetermined, e.g., by comparing the IC₅₀ of a compound in inhibiting oneor more of the types of T-type calcium channel with its IC₅₀ ininhibiting the one or more types of sodium channel: if the IC₅₀ forinhibiting the T-type calcium channels is lower than the IC₅₀ forinhibiting the sodium channel, the compound is considered selective. AnIC₅₀ ratio of 0.1 (or lower) denotes 10-fold (or greater) selectivity.An IC₅₀ ratio of 0.01 (or lower) denotes 100-fold (or greater)selectivity. An IC₅₀ ratio of 0.001 (or lower) denotes 1000-fold (orgreater) selectivity. In some embodiments, the selectivity for T-typecalcium channels is 10-fold or greater, 100-fold or greater, or1000-fold or greater.

The effectiveness of a compound in inhibiting T-type calcium channelsmay vary depending on the state of the T-type calcium channel that theT-type calcium channel antagonist inhibits. T-type calcium channels canoccur in different states depending on the cell membrane potential.T-type calcium channel antagonists that are effective in the methodsdescribed herein may include T-type calcium channel antagonists thatblock T-type calcium channels when the membrane potential is in therange from about −60 mV to about −30 mV, e.g., preferably about −40 mV.A membrane potential “in the range from about −60 to about −30 mV” caninclude membrane potentials within a range of −70 mV to −20 mV, orwithin a range of −65 mV to −25 mV, and can also encompass membranepotential ranges such as about −40 mV to about −30 mV, about −50 mV toabout −30 mV, about −70 mV to about −30 mV, about −50 mV to about −40mV, about −60 mV to about −40 mV, about −70 mV to about −40 mV, about−60 mV to about −50 mV, and about −70 to about −50 mV, as well as about−30 mV, about −40 mV, about −50 mV, and about −60 mV. In someembodiments, the T-type calcium channel antagonists that are effectivein the methods described herein may include T-type calcium channelantagonists that block T-type calcium channels when the membranepotential is in the range from about −100 mV to about −80 mV, e.g.,preferably about −90 mV. A membrane potential “in the range from about−100 to about −80 mV” can include membrane potentials within a range of−110 mV to −70 mV, or within a range of −105 mV to −75 mV, and can alsoencompass membrane potential ranges such as about −100 mV to about −80mV, about −90 mV to about −80 mV, and about −100 mV to about −90 mV, aswell as about −100 mV, about −90 mV, and about −80 mV.

While not being limited by any theory, it is believed that T-typecalcium channel antagonists that are effective in the methods describedherein may include T-type calcium channel antagonists that block T-typecalcium channels when the membrane potential is in the range from about−60 mV to about −30 mV, e.g., about −40 mV selectively when compared toblockade of the T-type calcium channels when the membrane potential isin the range from about −100 mV to about −80 mV, e.g., about −90 mV.

A T-type channel inhibitor that is effective may inhibit T-type calciumchannels with an IC₅₀ for inhibiting T-type calcium channels when themembrane potential is about −40 mV that is about 10 μM or lower, e.g.,about 1 μM or lower, about 500 nM or lower, about 100 nM or lower, about50 nM or lower, about 10 nM or lower, about 5 nM or lower, or about 1 nMor lower. A T-type calcium channel antagonist that is effective mayinhibit T-type calcium channels at a membrane potential of about −40 mVselectively compared to inhibition of T-type calcium channels at amembrane potential of about −90 mV. For example, the ratio of the IC₅₀of the T-type calcium channel antagonist in inhibiting T-type calciumchannels at a membrane potential of about −40 mV selectively compared toinhibition of T-type calcium channels at a membrane potential of about−90 mV may be about 1:2 or lower, e.g., about 1:5 or lower, about 1:10or lower, about 1:20 or lower, about 1:50 or lower, about 1:100 orlower, about 1:500 or lower, about 1:1000 or lower. In some embodiments,the selectivity for inhibiting T-type calcium channels at about −40 mVcompared to inhibiting T-type calcium channels at about −90 mV is 2-foldor greater, 5-fold or greater, 10-fold or greater, 100-fold or greater,or 1000-fold or greater.

A T-type channel inhibitor that is effective may inhibit T-type calciumchannels with an IC₅₀ for inhibiting T-type calcium channels when themembrane potential is about −90 mV that is about 10 μM or lower, e.g.,about 1 μM or lower, about 500 nM or lower, about 100 nM or lower, about50 nM or lower, about 10 nM or lower, about 5 nM or lower, or about 1 nMor lower. A T-type calcium channel antagonist that is effective mayinhibit T-type calcium channels at a membrane potential of about −90 mVselectively compared to inhibition of T-type calcium channels at amembrane potential of about −40 mV. For example, the ratio of the IC₅₀of the T-type calcium channel antagonist in inhibiting T-type calciumchannels at a membrane potential of about −90 mV selectively compared toinhibition of T-type calcium channels at a membrane potential of about−40 mV may be about 1:2 or lower, e.g., about 1:5 or lower, about 1:10or lower, about 1:20 or lower, about 1:50 or lower, about 1:100 orlower, about 1:500 or lower, about 1:1000 or lower. In some embodiments,the selectivity for inhibiting T-type calcium channels at about −90 mVcompared to inhibiting T-type calcium channels at about −40 mV is 2-foldor greater, 5-fold or greater, 10-fold or greater, 100-fold or greater,or 1000-fold or greater.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.hydrates and solvates) or can be isolated. In some embodiments, thecompounds provided herein, or pharmaceutically acceptable salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds providedherein. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds provided herein, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by converting an existing acid or base moiety to its salt form.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts of thepresent application include the conventional non-toxic salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. The pharmaceutically acceptable salts of the present applicationcan be synthesized from the parent compound which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g.,methanol, ethanol, iso-propanol, or butanol) or acetonitrile arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977).Methods for preparing salt forms are described, for example, in Handbookof Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH,2002.

IV. Combination Therapies

One or more additional therapeutic agents can be used in combinationwith the compounds provided herein for the treatment of AngelmanSyndrome or Prader-Willi syndrome. Example additional therapeutic agentsinclude, but are not limited to calcium channel antagonists (includingL-type and T-type antagonists), anticonvulsant agents, GABA(A) receptoragonists and positive allosteric modulators or gene therapy or genereactivation therapy (Bailus et al., The prospect of molecular therapyfor Angelman syndrome and other monogenic neurologic disorders, BMCNeuroscience, 2014, 15, 76).

In some embodiments, the treatment with the T-type calcium channelantagonist can be provided in the absence of additional pharmacologicalagents for treating Angelman Syndrome or Prader-Willi syndrome. In someembodiments, the treatment can be performed with a single T-type calciumchannel antagonist. In some embodiments, the treatment with the T-typecalcium channel antagonist can be provided in the absence of ananticonvulsant agent.

The one or more additional pharmaceutical agents can be administered toa patient simultaneously or sequentially, using the same schedule or adifferent schedule of administration, which will be determined by theparticular combination used and the judgment of the prescribingphysician.

Example calcium channel antagonists include, but are not limited to, theT-type calcium channel antagonists described herein, and L-type calciumchannel antagonists. In some embodiments, the additional calcium channelantagonist is selected from a T-type calcium channel antagonist providedherein. In some embodiments, the additional calcium channel antagonistis an L-type calcium channel antagonist. In some embodiments, theadditional calcium channel antagonist is a T-type calcium channelantagonist. In some embodiments, the additional calcium channelantagonist is a T-type calcium channel antagonist selected from thegroup consisting of mibefradil, MK-5395, MK-6526, MK-8998, and Z944. Insome embodiments, the additional calcium channel antagonist is a T-typecalcium channel antagonist and an L-type calcium channel antagonist. Insome embodiments, the additional calcium channel antagonist is a T-typecalcium channel antagonist or an L-type calcium channel antagonistselected from the group consisting of ACT-28077, mibefradil, andTTL-1177. In some embodiments, the additional calcium channel antagonistis mibefradil.

Example anticonvulsant agents include, but are not limited to,acetazolamide, carbamazepine, clobazam, clonazepam, eslicarbazepineacetate, ethosuximide, gabapentin, lacosamide, lamotrigine,levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam,phenobarbital, phenytoin, pregabalin, primidone, retigabine, rufinamide,valproate, e.g., sodium valproate, stiripentol, tiagabine, topiramate,vigabatrin, and zonisamide.

Example GABA(A) receptor agonists include gaboxadol, bamaluzole,gamma-aminobutyric acid, gabamide, gamma-amino-beta-hydroxybutyric acid,gaboxadol, ibotenic acid, isoguvacine, isonipecotic acid, muscimol,phenibut, picamilon, progabide, quisqualamine, SL 75102, andthiomuscimol.

Example GABA(A) receptor positive allosteric modulators includeavermectins (e.g., ivermectin), barbiturates (e.g., phenobarbital),benzodiazepines (e.g., adinazolam, alprazolam, bentazepam, bretazenil,bromazepam, brotizolam, camazepam, chlordiazepoxide, cinazepam,cinolazepam, clobazam, clonazepam, clonazolam, clorazepate, clotiazepam,cloxazolam, delorazepam, diazepam, diclazepam, estazolam, ethylcarfluzepate, etizolam, ethyl loflazepate, flubromazepam, flubromazolam,flunitrazepam, flurazepam, flutazolam, flutoprazepam, halazepam,ketazolam, loprazolam, lorazepam, lormetazepam, medazepam, mexazolam,midazolam, nifoxipam, nimetazepam, nitrazepam, nordiazepam, oxazepam,phenazepam, pinazepam, prazepam, premazepam, pyrazolam, quazepam,rilmazafone, temazepam, thienalprazolam, tetrazepam, and triazolam),bromides (e.g., potassium bromide, carbamates (e.g., meprobamate,carisoprodol), chloralose, chlormezanone, clomethiazole,dihydroergolines (e.g., ergoloid (dihydroergotoxine)), etazepine,etifoxine, Imidazoles (e.g., etomidate), kavalactones (found in kava),loreclezole, neuroactive steroids (e.g., allopregnanolone, ganaxolone),nonbenzodiazepines (e.g., zaleplon, zolpidem, zopiclone, eszopiclone),petrichloral, phenols (e.g., propofol), piperidinediones (e.g.,glutethimide, methyprylon), propanidid, pyrazolopyridines (e.g.,etazolate), quinazolinones (e.g., methaqualone), skullcap constituents,stiripentol, sulfonylalkanes (e.g., sulfonmethane, tetronal, trional),and valerian constituents (e.g., valeric acid, valerenic acid).

In some embodiments, the therapy can be administered as a monotherapy.In some embodiments, the therapy can be administered in the absence ofadditional antiepileptic therapy. The therapy can be administered in theabsence of any of the additional agents described in this section. Forexample, the therapy can be administered in the absence of additionalanticonvulsant such as, acetazolamide, carbamazepine, clobazam,clonazepam, eslicarbazepine acetate, ethosuximide, gabapentin,lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine,perampanel, piracetam, phenobarbital, phenytoin, pregabalin, primidone,retigabine, rufinamide, valproate, e.g., sodium valproate, stiripentol,tiagabine, topiramate, vigabatrin, or zonisamide.

Example gene reactivation therapies include administration of compoundsthat activate the paternal (or maternal) copy of UBE3A. Examples includetopoisomerase inhibitors (including topoisomerase I and II inhibitors)such as topotecan, irinotecan, etoposide and dexrazoxane, and othercompounds identified by Huang et al., “Topoisomerase inhibitorsunsilence the dormant allele of Ube3a in neurons”, Nature, 2011, 481,185-89.

In some embodiments, the T-type calcium channel antagonists providedherein can be used in combination with one or more additional therapiesincluding, but not limited to, physical therapy, occupational therapy,communication therapy, and behavioral therapy. In some embodiments, theT-type calcium channel antagonists provided herein can be used incombination with one or more additional therapeutic agents and one ormore additional therapies selected from the group consisting of physicaltherapy, occupational therapy, communication therapy, and behavioraltherapy.

V. Pharmaceutical Compositions

The T-type calcium channel inhibitors used in the methods describedherein can be administered in the form of pharmaceutical compositions.Thus the present disclosure provides T-type calcium channel inhibitor,and at least one pharmaceutically acceptable carrier for use in theclaimed methods of treatment, or the manufacture of a medicament fortreating conditions as described herein. These compositions can beprepared in a manner known in the pharmaceutical art, and can beadministered by a variety of routes. Administration may be topical(including transdermal, epidermal, ophthalmic and to mucous membranesincluding intranasal, vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be,e.g., by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

This pharmaceutical compositions which contain, as the activeingredient, a T-type calcium channel inhibitor (which can be in the formof a pharmaceutically acceptable salt), in combination with one or morepharmaceutically acceptable carriers (excipients). In some embodiments,the composition is suitable for topical administration. In making thecompositions of the invention, the active ingredient is typically mixedwith an excipient, diluted by an excipient or enclosed within such acarrier in the form of, e.g., a capsule, sachet, paper, or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, e.g., up to 10% by weight of theactive compound, soft and hard gelatin capsules, suppositories, sterileinjectable solutions and sterile packaged powders.

In preparing a formulation, the T-type calcium channel inhibitor can bemilled to provide the appropriate particle size prior to combining withthe other ingredients. If the active compound is substantiallyinsoluble, it can be milled to a particle size of less than 200 mesh. Ifthe active compound is substantially water soluble, the particle sizecan be adjusted by milling to provide a substantially uniformdistribution in the formulation, e.g., about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other Formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. TheFormulations can additionally include: lubricating agents such as talc,magnesium stearate and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents.

In some embodiments, the pharmaceutical composition comprises silicifiedmicrocrystalline cellulose (SMCC) and at least one compound describedherein, or a pharmaceutically acceptable salt thereof. In someembodiments, the silicified microcrystalline cellulose comprises about98% microcrystalline cellulose and about 2% silicon dioxide wt/wt.

In some embodiments, a wet granulation process is used to produce thecomposition. In some embodiments, a dry granulation process is used toproduce the composition.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1,000 mg (1 g), more usually about 100mg to about 500 mg, of the active ingredient. In some embodiments, eachdosage contains about 10 mg of the active ingredient. In someembodiments, each dosage contains about 50 mg of the active ingredient.In some embodiments, each dosage contains about 25 mg of the activeingredient. The term “unit dosage forms” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient.

The components used to formulate the pharmaceutical compositions are ofhigh purity and are substantially free of potentially harmfulcontaminants (e.g., at least National Food grade, generally at leastanalytical grade, and more typically at least pharmaceutical grade).Particularly for human consumption, the composition is preferablymanufactured or Formulated under Good Manufacturing Practice standardsas defined in the applicable regulations of the U.S. Food and DrugAdministration. For example, suitable Formulations may be sterile and/orsubstantially isotonic and/or in full compliance with all GoodManufacturing Practice regulations of the U.S. Food and DrugAdministration.

The active compound may be effective over a wide dosage range and isgenerally administered in a therapeutically effective amount. It will beunderstood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms and the like.

The therapeutic dosage of a compound of the present invention can varyaccording to, e.g., the particular use for which the treatment is made,the manner of administration of the compound, the health and conditionof the patient, and the judgment of the prescribing physician. Theproportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 μg/kg to about1 g/kg of body weight per day. In some embodiments, the dose range isfrom about 0.01 mg/kg to about 100 mg/kg of body weight per day. Thedosage is likely to depend on such variables severity of the disease,the overall health status of the particular patient, the relativebiological efficacy of the compound selected, Formulation of theexcipient, and its route of administration. Effective doses can beextrapolated from dose-response curves derived from in vitro or animalmodel test systems. Effective doses for a human can be, e.g., about 1mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg,50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg,100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg,180 mg, 190 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg,700 mg, 800 mg, 900 mg or 1000 mg. The doses can be administered, e.g.,once a day, twice a day, three times a day, or four times a day.

In some embodiments, when the T-type calcium channel antagonist ismibefradil, and the mibefradil can be administered at a dose of, e.g.,about 0.1 mg, 0.3 mg, 1 mg, 3 mg, 5 mg, 10 mg. 15 mg. or 30 mg. Thedoses can be administered, e.g., once a day, twice a day, three times aday, or four times a day.

In some embodiments, when the T-type calcium channel antagonist isMK-5395, and the MK-5395 can be administered at a dose of, e.g., about0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg.The doses can be administered, e.g., once a day, twice a day, threetimes a day, or four times a day.

In some embodiments, the T-type calcium channel antagonist is MK-6526,and the MK-6526 can be administered at a dose of, e.g., about 0.3 mg/kg,1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The dosescan be administered, e.g., once a day, twice a day, three times a day,or four times a day.

In some embodiments, the T-type calcium channel antagonist is MK-8998,and the MK-8998 can be administered at a dose of, e.g., about 0.3 mg/kg,1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The dosescan be administered, e.g., once a day, twice a day, three times a day,or four times a day.

In some embodiments, the T-type calcium channel antagonist is Z944, andthe Z944 can be administered at a dose of, e.g., about 0.3 mg/kg, 1mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg. 30 mg/kg, or 100 mg/kg. The doses canbe administered, e.g., once a day, twice a day, three times a day, orfour times a day.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, e.g., about 0.1 to about 1000 mg of the activeingredient of the present invention.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face mask, tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theFormulation in an appropriate manner.

Topical formulations can contain one or more carriers. In someembodiments, ointments can contain water and one or more hydrophobiccarriers selected from, e.g., liquid paraffin, polyoxyethylene alkylether, propylene glycol, white petroleum jelly, and the like. Carriercompositions of creams can be based on water in combination withglycerol and one or more other components, e.g., glycerinemonostearate,PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can beformulated using isopropyl alcohol and water, suitably in combinationwith other components such as, e.g., glycerol, hydroxyethyl cellulose,and the like. In some embodiments, topical formulations contain at leastabout 0.1, at least about 0.25, at least about 0.5, at least about 1, atleast about 2 or at least about 5 wt % of the compound of the invention.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to eliminate or atleast partially alleviate the symptoms of the disease and itscomplications. Effective doses will depend on the disease conditionbeing treated as well as by the judgment of the attending cliniciandepending upon factors such as the severity of the disease, the age,weight and general condition of the patient and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers or stabilizers will resultin the formation of pharmaceutical salts.

The therapeutic dosage of a T-type calcium channel antagonist used inthe methods described herein can vary according to, e.g., the particularuse for which the treatment is made, the manner of administration of thecompound, the health and condition of the patient, and the judgment ofthe prescribing physician. The proportion or concentration of a compoundof the invention in a pharmaceutical composition can vary depending upona number of factors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, theT-type calcium channel antagonists can be provided in an aqueousphysiological buffer solution containing about 0.1 to about 10% w/v ofthe compound for parenteral administration. Some typical dose ranges arefrom about 1 μg/kg to about 1 g/kg of body weight per day. In someembodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kgof body weight per day. The dosage is likely to depend on such variablesas the type and extent of progression of the disease or disorder, theoverall health status of the particular patient, the relative biologicalefficacy of the compound selected, formulation of the excipient, and itsroute of administration. Effective doses can be extrapolated fromdose-response curves derived from in vitro or in vivo model testsystems.

EXAMPLES

The invention is further described in the following example, which doesnot limit the scope of the invention defined in the claims.

Example 1. Treatment of a Patient Having Angelman Syndrome

A patient has Angelman Syndrome, which is characterized by mentaldisability and frequent erratic movement (e.g., seizures,hand-flapping). The patient is administered, alone or in combinationwith other therapies, a therapeutically effective amount of a T-typecalcium channel antagonist provided herein (e.g., mibefradil,efonidipine, TTL-1177, nickel, and the like). After a sufficient dosageof the T-type calcium channel antagonist has been administered (e.g.,after one dose or after a series of doses), the patient's brain activityis monitored (e.g., using an electroencephalogram (EEG)). The patient'sbrain activity demonstrates measurable improvement in AngelmanSyndrome-characteristic brain activity compared to the brain activity ofthe patient prior to treatment with the T-type calcium channelantagonist. The patient may also exhibit improvements in other symptoms,including intellectual and developmental disability, sleep disturbance,seizures, and jerky movements.

Example 2. In Vivo Study in Murine Angelman Model

Three separate tests were performed to determine effects of twocompounds, mibefradil and MK-8998 on behavioral and physiologicaldeficits and epilepsy in a mouse model for Angelman syndrome. The testsincluded: audiogenic-induced epilepsy test (Test 1, T1), a battery ofmotor/behavioral tests (Test 2, T2) and long-term potentiationmeasurements (LTP, Test 3, T3). Completion of each test provided aquantitative result of mibefradil or MK-8998 effect on this test. Forall three tests, 2 separate groups of mice were generated: group 1 fortest 1 and group 2 for tests 2 and 3, as shown below in see Table 2 andeach group consists of 6 subgroups, a-f.

For all behavioral and electrophysiology experiments female Ube3am⁻/p⁺KOmice (i.e., AS mice) were crossed with wild-type males, to generateheterozygous AS mice and littermate controls in the F1 hybrid12952-C57BL/6 background. For epilepsy tests, AS mice are crossed withcontrols in the 12952/SvPasCrl background. In total, 2 groups of miceare required: group 1 for the epilepsy test (Table 2, group 1a-f) andgroup 2 for the behavioral test battery and electrophysiology (Table 2,group 2a-f). Each group consists of 6 subgroups, including 2 controlsubgroups: wild-type (a) and AS mutants (b) mice on standard diet, and 4subgroups on drug-supplemented diets: Mibefradil (c-d) and MK-8998 (e-f)groups each at two different concentrations. The number of mice used pertest/subgroup is indicated in Table 2 and ranges from 5-7 mice/subgroupfor electrophysiology, 10 mice/subgroup for epilepsy test, and 12 to 15mice/subgroup for behavioral test battery. Adult male and female mice(age <8 weeks) for were used for all experiments.

TABLE 2 Group/ N/ Model/ Test subgroup group subgroup Treatment T1 1a 10Wildtype Standard chow T1 1b 10 Ube3a^(stop/p+) Standard chow T1 1c 10Ube3a^(stop/p+) Mibefradil T1 1d 10 Ube3a^(stop/p+) Mibefradil T1 1e 10Ube3a^(stop/p+) CX-8998 T1 1f 10 Ube3a^(stop/p+) CX-8998 T2-3* 2a 12-15** Wildtype Standard chow T2-3 2b 12-15 Ube3a^(stop/p+) Standardchow T2-3 2c 12-15 Ube3a^(stop/p+) Mibefradil T2-3 2d 12-15Ube3a^(stop/p+) Mibefradil T2-3 2e 12-15 Ube3a^(stop/p+) CX-8998 T2-3 2f12-15 Ube3a^(stop/p+) CX-8998 *Test 2 performed in two batches of 7-8mice/subgroup/batch. **for behavioral test battery: at minimum 12mice/subgroup tested and 15 mice/subgroup for statistical power.

Mibefradil and MK-8998 were tested at various different doses (e.g., 10mg/kg, 30 mg/kg, 40 mg/kg, 60 mg/kg) based on the assumption that miceeat 5 g chow/day. Both drugs were administered in chow starting afterthe weaning of the pups (P21; subgroups c-f). Control mice (subgroup aand b) received standard chow.

Effect of Mibefradil and MK-8998 on Epilepsy in Angelman Syndrome (AS)Mouse Model (Test 1)

AS-mutant mice, wild-type, and AS mutant mice were maintained on eitherstandard chow (group 1a and Ib), or on drug-supplemented chow (groupIc-f) from postnatal day 21 (P21) until adulthood. At age of 8 weeks(P56) all mice were tested for occurrence of audiogenic-inducedseizures. The outcome of this test provided a number (or %) ofmice/subgroup which experienced epilepsy. Most (>90%) AS-mutant mice onstandard diet (group Ib) were expected to experience epilepsy. Incontrast, wild-type mice on standard diet (group 1a) were not expectedto show seizures (<15%) (see e.g., Silva-santos et al., J. Clin.Invest., 2015, 125(5), 2069-2076). A brief summary of the Test 1protocol is shown below:

1. Epilepsy Assessment: (12952/SvPasCrl×AS Mice)

a. 9 mice/group

b. 6 groups as follows:

-   -   i. Vehicle: Wild type    -   ii. Mibefradil (Oral gavage)        -   1. 10 mg/kg        -   2. 40 mg/kg    -   iii. MK-8998 (Oral gavage)        -   1. 10 mg/kg        -   2. 30 mg/kg        -   3. 60 mg/kg

c. Treatment schedule: Baseline epilepsy test→Begin 7 days of treatment,P56→Epilepsy test

d. Results: % of mice to experience audiogenic-induced epilepsy

First, the mice underwent the epilepsy test prior to treatment with themibefradil or MK-8998. The mice were then treated with either mibefradilor MK-8998 via oral gavage for 7 days and subsequently underwent theepilepsy test. The mice were dosed as follows: Group 1: mibefradil 10mg/kg and 40 mg/kg; MK-8998: 10 mg/kg and 30 mg/kg. Group 2: mibefradil10 mg/kg and 40 mg/kg; MK-8998: 30 mg/kg and 60 mg/kg. Results of theaudiogenic-induced epilepsy tests are shown in FIG. 1.

The results show that, remarkably, MK-8998 eliminated seizures in adose-dependent manner, with seizures being eliminated completely inabout 80% of treated animals at 60 mg/kg. It is important to note,however, that the absence of an apparent response in the data shown inFIG. 1 does not show that treatment was ineffective since FIG. 1 onlyrecords the number of seizure free animals recorded. An animal couldhave a reduced number of seizures or reduced severity of seizures, yetstill be recorded as having seizures in the data shown in FIG. 1.

It is expected that the seizure-prone animals treated with mibefradil orMK-8998 or other T-type calcium channel inhibitor exhibit a reducednumber of seizures, or a reduced severity of seizures, or both.

Test 2. Effect of Mibefradil and MK-8998 on Behavioral Deficits inAS-Mice

A battery of well-established motor coordination and behavioral tests inwhich AS-mutant mice show a robust phenotype (Silva-santos et al., J.Clin. Invest., 2015, 125(5), 2069-2076) and which includes rotarod, openfield, marble burying, nest building and forced swim test will becarried out. For this tests battery a group consisting of 12-15mice/subgroup (6 subgroups, see Table 2) will be tested resulting in agroup of 72-90 mice. Due to limitations in breeding and testingcapacity, this group will be split into two batches consisting of 7-8mice/subgroup (42-48 mice/batch). Mice from both batches will undergothe same battery of motor/behavioral tests and results from both groupswill be combined to reach statistical power. After each test (e.g.rotarod) a quantifiable result (outcome measure) will be obtain for eachsubgroup. A brief summary of the Test 2 protocol is shown below:

2. Behavioral Assessment: Heterozygous AS Mice (Male Wild Type×FemaleUbe3am−/p+KO Mice)

a. 12-15 mice/group

b. 6 groups as follows:

-   -   i. Vehicle: Wild type    -   ii. Vehicle: Heterozygous AS mouse    -   iii. Mibefradil        -   1. 10 mg/kg        -   2. 40 mg/kg    -   iv. MK-8998        -   1. 30 mg/kg        -   2. 60 mg/kg

c. Treatment schedule: Testing completed in 2 batches of 6 weeks oftesting

d. Assessments and Results:

-   -   i. Accelerating Rotarod: Latency (s)    -   ii. Marble Burying: Number of Marbles Unburied    -   iii. Open Field Test: Path length (m)    -   iv. Nest Building: material used (%)    -   v. Forced Swim Test: Floating time (%).

Test 3. Effect of Mibefradil and MK-8998 on Synoptic Plasticity Deficitsin AS Mice.

Subsequently, the effect of mibefradil and MK-8998 on long termpotentiation (LTP) deficit in AS-mutant mice will be tested. For thisexperiments, mice which underwent Test 2 can be used. LTP will bemeasured in 5-7 mice/subgroup. If required, a higher number of mice canbe tested (e.g., from the second batch of mice) A brief summary of theTest 3 protocol is shown below:

3. Long Term Potentiation Assessment: Heterozygous AS Mice (Male WildType×Female Ube3am−/p+KO Mice)

a. 6-7 mice/group

b. 6 groups as follows:

-   -   i. Vehicle: Wild type    -   ii. Vehicle: Heterozygous AS mouse    -   iii. Mibefradil        -   1. 10 mg/kg        -   2. 40 mg/kg    -   iv. MK-8998        -   1. 30 mg/kg        -   2. 60 mg/kg

c. Treatment schedule: 2 mice per day. 5 weeks total

d. Results:

-   -   i. LTP induction and expression (% of baseline) in wild-type        mice    -   ii. Potential of a drug to rescue LTP-deficit in AS mice (% of        baseline)

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Anumber of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other aspects, advantages, embodiments and modificationsare within the scope of the following claims.

1. A method of treating Angelman Syndrome or Prader-Willi syndrome,comprising administering to a subject in need of such treatment atherapeutically effective amount of a T-type calcium channel antagonist.2. The method of claim 1, wherein the T-type calcium channel antagonistis a calcium channel antagonist that selectively targets T-type calciumchannels Cav3.1, Cav3.2, and/or Cav3.3.
 3. The method of claim 1,wherein the T-type calcium channel antagonist is a small molecule, anantibody, or a siRNA. 4-8. (canceled)
 9. The method of claim 1, whereinthe T-type calcium channel antagonists antagonize a T-type calciumchannel in a cell when the membrane potential of the cell is in therange from about −60 mV to about −30 mV, e.g., about −40 mV.
 10. Themethod of claim 1, wherein said T-type calcium channel antagonist isselected from the group consisting of mibefradil, MK-8998, diltiazem,nifedipine, nitrendipine, nimodipine, niludipine, niguldipine,nicardipine, nisoldipine, amlodipine, felodipine, isradipine, ryosidine,gallopamil, verapamil, tiapamil, pimozide, thioridazine, NNC 55-0396,TTL-1177, anandamide, pimozide, penfluridol, clopimozide, fluspirilene,haloperidol, droperidol, benperidol, triperidol, melperone, lenperone,azaperone, domperidone, antrafenine, aripiprazole, ciprofloxacin,dapiprazole, dropropizine, etoperidone, itraconazole, ketoconazole,levodropropizine, mepiprazole, naftopidil, nefazodone, niaprazine,oxypertine, posaconazole, trazodone, urpidil, vesnarinone, manidipine,nilvadipine, benidipine, efonidipine, flunarizine, anandamide,lomerizine, phenytoin, zonisamide, U-92032, tetralol, mibefradil, NNC55-0396, TTA-A2, TTA-A8, TTA-P1, 4-aminomethyl-4-fluoropiperidine(TTA-P2), TTA-Q3, TTA-Q6, MK-5395, MK-6526, MK-8998, Z941, Z944,phensuximide, mesuximide, desmethylmethsuximide, efonidipine,trimethadione, dimethadione, ABT-639, KYSO5044, nickel, and kurtoxin,and combinations thereof. 11-14. (canceled)
 15. The method of claim 1,wherein the treatment comprises reducing or ameliorating at least oneneurological symptom in the subject, wherein neurological symptomcomprises one or more of hyperactivity, abnormal sleep pattern, seizure,dystonia and ataxia.
 16. (canceled)
 17. The method of claim 15, whereinthe neurological symptom comprises seizure, and wherein the treatmentcomprises reducing the frequency of seizure in the subject and/orreducing the severity of seizure in the subject.
 18. (canceled)
 19. Themethod of claim 15, wherein the neurological symptom comprises dystonia,and wherein the treatment comprises reducing the frequency of dystoniain the subject and/or the severity of dystonia in the subject. 20.(canceled)
 21. The method of claim 15, wherein the neurological symptomcomprises ataxia, and wherein the treatment comprises reducing thefrequency of ataxia in the subject and/or the severity of ataxia in thesubject.
 22. (canceled)
 23. The method of claim 1, wherein the treatmentcomprises one or more of improving cognition or reducing cognitivedeficits in the subject, improving memory or reducing memory deficits inthe subject, improving attention or reducing attention deficits in thesubject, reducing the amount or rate of weight gain in the subject,and/or reducing hunger or increasing satiety in the subject. 24-29.(canceled)
 30. The method of claim 1, wherein said T-type calciumchannel antagonist substantially crosses the blood brain barrier. 31.The method of claim 1, wherein said T-type calcium channel agonist doesnot substantially cross the blood brain barrier.
 32. The method of claim1, further comprising administering to the subject an additionaltherapeutic agent.
 33. The method of claim 32, wherein the additionaltherapeutic agent is an additional T-type calcium channel inhibitor. 34.The method of claim 32, wherein the additional therapeutic agent is ananticonvulsive agent.
 35. The method of claim 1, further comprisingadministering an additional therapy selected from the group consistingof physical therapy, occupational therapy, communication therapy, andbehavioral therapy.
 36. A method of treating a disease or disorderassociated with CaMKII autophosphorylation in a subject in need thereof,comprising administering to the subject a T-type calcium channelantagonist.
 37. The method of claim 36, wherein the disease or disorderis Angelman Syndrome or Prader-Willi syndrome.
 38. (canceled)
 39. Themethod of claim 36, wherein the T-type calcium channel antagonist is acalcium channel antagonist that selectively targets T-type calciumchannels Cav3.1, Cav3.2, and/or Cav3.3.
 40. The method of claim 36,wherein the T-type calcium channel antagonist is a small molecule, anantibody, or a siRNA. 41-45. (canceled)
 46. The method of claim 36,wherein the T-type calcium channel antagonists antagonize a T-typecalcium channel in a cell when the membrane potential of the cell is inthe range from about −60 mV to about −30 mV, e.g., about −40 mV.
 47. Amethod of treating obesity in a subject in need thereof, comprisingadministering to the subject a T-type calcium channel antagonist. 48.The method of claim 47, wherein the treatment comprises reducing bodyweight in the subject.
 49. The method of claim 47, wherein the treatmentcomprises reducing the amount or rate of weight gain in the subjectand/or reducing hunger or increasing satiety in the subject. 50-51.(canceled)
 52. The method of claim 47, wherein the T-type calciumchannel antagonist is a calcium channel antagonist that selectivelytargets T-type calcium channels Cav3.1, Cav3.2, and/or Cav3.3.
 53. Themethod of claim 47, wherein the T-type calcium channel antagonist is asmall molecule, an antibody, or a siRNA. 54-58. (canceled)
 59. Themethod of claim 47, wherein the T-type calcium channel antagonistsantagonize a T-type calcium channel in a cell when the membranepotential of the cell is in the range from about −60 mV to about −30 mV,e.g., about −40 mV.